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| The EMBO Journal
(1997) 16, 6325–6336, doi:10.1093/emboj/16.21.6325 |
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| Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination |
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| O. Staub1, I. Gautschi2, T. Ishikawa1, K. Breitschopf3, A. Ciechanover3, L. Schild2 and D. Rotin1 |
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1 The Hospital for Sick Children, Division of Respiratory Research, 555 University Avenue, Toronto, Ontario M5G 1X8 and Biochemistry Department, University of Toronto, Toronto, Canada 2 Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1015 Lausanne, Switzerland 3 Biochemistry Department, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel To whom correspondence should be addressed D. Rotin, drotin@sickkids.on.ca Received 23 April 1997; Revised 11 August 1997. |
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| Abstract |
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The epithelial Na+ channel (ENaC), composed of three subunits (   ), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short-lived protein (t1/2  1 h) that is ubiquitinated in vivo on the and (but not ) subunits. Mutation of a cluster of Lys residues (to Arg) at the N-terminus of ENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N-terminal Lys to Arg mutations in ENaC, but not in ENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell-surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled ENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination-mediated regulation of ion channels. |
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| Keywords: ENaC, half-Life, lysosome, proteasome, ubiquitination |
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